FR2739028A1 - IMPROVED CELL ENCAPSULATION DEVICE - Google Patents

Abstract

The present invention is directed to a cell encapsulation device that permits rapid and straightforward cell transfer into the device. The preferred device includes components that allow a user to quickly transfer cells into the device with minimal risk to the cells. Among the most important improvements of the present invention are: automatic filtration of excess solution during cell transfer; an instantly wettable cover, allowing ready view into the cell chamber; and a swellable core, allowing cells to be transferred with minimal shear force while assuring optimal cell placement in the device during use. The device of the present invention may be used either in vivo, such as to deliver therapeutic substances, or in vitro, such as to serve as bioreactor.

Description

IMPROVED CELL ENCAPSULATING DEVICE
The present invention relates to devices used to encapsulate living cells and, in particular, to cell encapsulating devices designed to maintain the viability of the cells placed therein.

 Various devices for encapsulating cells have been described in recent years. These devices are often used to deliver therapeutic substances to a recipient or as a bioreactor. The devices usually come in one of two forms: microencapsulation devices or macroencapsulation devices.

 In a microencapsulation device, small amounts of cells are usually suspended in a small drop and enclosed in a semi-permeable membrane. The semipermeable membrane allows, for example, nutrients, waste and therapeutic agents, to diffuse through the membrane, while preventing, for example, cells and antibodies, from migrating through the membrane. In order to provide enough cells to achieve the desired result, a large number of microencapsulation devices are usually used.

 A common limitation of microencapsulation devices is the instability of the microcapsule membrane once it is implanted in a recipient or placed in a bioreactor. Such instability often leads to cell death or inconsistent release of therapeutic agents. This may result in the obligation to proceed with the obligation to carry out more than one administration of microcapsules.

 Another limitation in the number of such devices is the possibility of an immunogenic reaction of the recipient to the composition used to make the semi-permeable membrane of the device. This can lead to a serious condition of a recipient and / or damage to the device.

 An additional limitation is the difficulty of removing the miaocapsules from a recipient or a bioreactor.

As representative examples of microencapsulation devices, there may be mentioned, without limitation, those of US patents No. 5,182,111, 5,283,187, and 5,389,535, all issued to Aebischer et al., Those of US patents No. 4,487,758.4 673,566.4 689,293, 4,806,355, and 4,897,758, all issued to Goosen et al., Of U.S. Patent No. 4,803,168, issued to
Jarvis Jr., U.S. Patent Nos. 4,352,883 and 4,391,909, both issued to
Lim, US Patent No. 4,298,002, issued to Ronel et al., And US Patent

 NO 4 353 888, issued to Sefton.

 In a macroencapsulation device, more cells are enclosed in a chamber of a certain type. These devices have at least one semi-permeable membrane to allow the necessary flow of fluids, while retaining the cells securely. As representative examples of macroencapsulation devices, non-limiting mention may be made of the devices of U.S. Patent Nos. 5,262,055, issued to Bae et al., Of the U.S. Patent

No. 4,911,717, issued to Gaskill, m, of US Patent No. 4,298,002, issued to
Ronel et al., From US Patent No. 5,387,237, issued to Fournier et al., From patent application PCT / AU90 / 00281, filed by Baxter International
Inc., from US Patent No. 5,413,471, issued to Brauker et al., From US Patent No. 5,344,454, issued to Clarke et al., From US Patent No. 5,002,661, issued to
Chick et al., And patent application PCT / US94 / 07190, filed by WL
Gore & Associates, Inc.

 A particularly advantageous macroencapsulation device is that described in application PCT / US94 / 07190, filed by W.L. Gore & Associates, Inc., which is incorporated here by reference (and hereinafter called "Gore device"). The Gore device is preferably an encapsulation device, of generally cylindrical shape comprising a flexible core displacing cells contained in a selectively permeable membrane. The selectively permeable membrane retains the cells inside the device while allowing the exchange of biochemicals between the encapsulated cells and the external surface of the device. In the case where the cell encapsulation device is inserted in a recipient and contains allogenic or xenogenic cells, the selectively permeable membrane also serves to isolate encapsulated cells from the recipient's immune system. The selective permeability of the membrane can be adjusted by impregnating the membrane with an appropriate hydrogel. The cell displacement nucleus positions the encapsulated cells near the selectively permeable membrane. In this way, the nucleus positions the cells encapsulated in the device, at a distance from a source of nutrients and with a cell density which minimizes the diffusion distance that the biochemical substances must cross between each encapsulated cell and the external environment of the device. This configuration makes it possible to maintain a maximum number of cells encapsulated in a given volume, at high levels of viability and productivity. During assembly of the device, the cells are introduced into the device in the form of a suspension, through an open end of the device.

The open end is then hermetically closed.

 In the biotechnology and pharmaceutical industries, for example, there is a need to quickly and easily screen suspected therapeutic agents, produced by cells, for toxicity, bioactivity, efficacy, and other factors . Conventional techniques for screening for therapeutic agents produced by cells include culturing the cells in vitro until sufficient quantities of cells are obtained to produce enough therapeutic agent for screening. Once the desired population of cells has been established, the cells are allowed to secrete their products into the culture medium until sufficient amounts of the therapeutic agent are produced. The culture medium containing the suspected therapeutic agent is then separated cells and concentrated, if necessary. The presumed therapeutic agent is usually extracted from the culture medium and purified before screening. The purified agent is then screened in vitro and / or in vivo. This is often a laborious, expensive and time consuming process.

 A device and method which would eliminate some of the steps of conventional screening techniques would be very useful. Although the Gore device, supra, is particularly suitable for this type of screening, further refinements to this device are considered possible.

 One method of eliminating some of the steps of screening for suspected therapeutic agents would be to implant a cell encapsulation device of the present invention, containing cells which produce the therapeutic agent, in a test subject. Once the cell encapsulation device has been implanted in a test subject, the presumed therapeutic agent will be administered directly to the subject, eliminating the need to separate the cells from a culture medium and to concentrate, purify and to administer the agent. Various tests for the suspected agent can be performed on the test subject or on samples taken from it to assess the therapeutic agent.

 In the field of gene therapy, for example, one method for performing a desired therapy is to collect certain cells from a patient or donor and genetically manipulate the cells ex vivo to express a gene product. The gene product is often a substance that the patient needs, but is not produced, or is inappropriately produced by the patient's own cells.

Once the cells have been modified by genetic engineering to produce the desired gene product, the cells are introduced directly into the patient with the idea that the cells will survive in the patient and produce the gene product in quantities and for a period of time sufficient to correct or improve the gene product deficiency in the patient. Since the genetically modified cells are introduced directly into the patient, the cells are essentially free to move and migrate throughout the patient's body. This poses a serious difficulty because cells modified by genetic engineering are often transformed and contain oncogenes. The presence of such transformed mobile cells in a patient often presents an unacceptable risk to the health of the patient.

 An implantable cell encapsulation device that prevents genetically engineered cells from coming into contact with cells of a patient and from migrating through the patient's tissues while releasing a therapeutic gene product in the patient from the cells encapsulated would be useful in the field of gene therapy.

 The present invention is a cell encapsulation device improved to contain living cells and maintain their viability. Although the device of the present invention has a wide range of applications, it is particularly suitable for the rapid testing of suspected therapeutic substances or agents produced by cells.

The device is also particularly suitable for containing genetically engineered cells while allowing a desired gene product produced by the encapsulated cells to be supplied by the cells to a patient or to a tissue culture. An embodiment of the present invention comprises: a selectively permeable shell comprising a microporous material; a core in the shell, the core preferably comprising a swellable material which expands from a first initial radial dimension to a second enlarged radial dimension after exposure to an aqueous solution; at least one opening in the envelope; a cell delivery device for transferring cells into the device through the opening; and means for hermetically closing the opening after introduction of the cells into the device.

 This structure allows the rapid and easy introduction of cells into the device, with a minimum shear force applied to the cells, the preferred inflatable nucleus gradually positioning the cells in a correct functional position after introduction of the cells into the device. Ideally, the sealing means of the device acts automatically, once the device has been filled with cells and the cell delivery device has been removed.

 In a still preferred embodiment of the present invention, the cell encapsulation device comprises an external envelope sufficiently permeable to water to allow rapid separation of the cells and the water after introduction of the cells in suspension in the device under relatively low pressures. This allows easy concentration of cells in the device without preconcentration of the cells.

In the accompanying drawings:
- Figure 1 illustrates a sectional view of the device 10 of the present invention comprising a core 11 inside an envelope 12 closed in leaktight manner against an optional spacer 18 with a sealing means 16. The optional spacer 18 is placed on the core 11 to facilitate the sealed closure of the device. The space between the envelope 12 and the nucleus 11 is called the cell zone 14, intended to contain cells. A cell distribution device 19 is also shown in FIG. 1. The point "A" in FIG. 1 indicates the general surface in which there is optionally a cut in the device 10 to remove the part of the device containing the spacer 18 , with the corresponding part of the casing 12, and the corresponding part of the core 11.

 FIG. 2 represents a sectional view of the device 20 of the present invention with an envelope 22 having an open end 25 and a closed end 27. The envelope 22 defines a zone of cells 24 between the envelope 22 and the core 21. The core 21 is fixed in the closed end of the device with a sealing means 26. A cell distribution device 29 is provided and constitutes a component of the device.

 FIG. 3 represents the device of FIG. 1 assembled with a syringe 33 drawn in section containing cells 37 to be loaded into the device 30 through a cell distribution device 39 fixed to the assembly. The core 31, the casing 32, the sealing means 36, and the spacer 38 are similar to the components 11, 12, 16, and 18, respectively in FIG. 1.

 FIG. 4 represents the assembly of FIG. 1 comprising a zone of cells 44 situated between the core 41 and the envelope 42 loaded with cells 47, closed by a sealing means 46, and separated from the cell distribution device 49. FIG. 4 also represents the part of the device containing the spacer 48, the sealing means 46, as well as the corresponding part of the casing 42, and the corresponding part of the core 41 having been removed from the device.

 FIG. 5 illustrates a method of loading a device of the present invention 50 with cells 57 in the cell area 54 located between the core 51 and the envelope 52 through the cell distribution device 59 connected to a source 53 cells (for example a syringe). A sealing means 56 and a spacer 58 are also shown. In the figure, a small amount of fluid 55 from the cell suspension is shown as having filtered through the envelope 52, having collected on the surface of the envelope 52, and having started to drip from the 'envelope 52.

 FIG. 6 represents a sectional view of a device 60 of the present invention with a core 61 comprising a spacer 63 made in one piece with that, comprising additional material at one end of the core to replace or complete the spacer separated 18 shown in Figure 1. The casing 62, the cell area 64, the sealing means 66, the cell distribution device 69 are also shown in the figure.

 FIG. 7 represents a sectional view of a device of the present invention 70 with a core 71 having spacers in one piece with it, 73 and 75, in order to replace or complete the spacer 18 shown in FIG. 1. In this embodiment, a cell distribution device 79 is placed adjacent to the core 71 and held in place with a sealing means 76. The envelope 72, the cell zone 74, and the cell delivery device, 79, are also shown in the figure.

 Referring now to Figure 1, the device 10 of the present invention comprises a core 11 located inside a casing 12 which is sealed with a sealing means 16. Between the core and the envelope is a cell area 14 capable of containing cells. A spacer 18 is optionally provided to help maintain the dimensions of the cell area 14 and facilitate sealing of the device. In addition, a cell distribution device 19 is provided as a component of the device. After removal of the cell delivery device 19 from inside the cell area 14, the sealing means 16 contracts to seal the device and encapsulate the cells contained therein.

 The present invention relates to a cell encapsulation device which can be used either in vivo or in vitro. Used for example in vivo, the present invention can release therapeutic substances in a recipient, from cells encapsulated in a device implanted in the recipient. The therapeutic substance can be derived from cells similar to cells absent or deficient in the recipient. Alternatively, the therapeutic substance can be derived from genetically engineered cells to produce gene products that are absent or inappropriately produced by the recipient's own cells.

 For in vitro use, the present invention, with a supply of cells encapsulated in the device, can function as a bioreactor, for example.

 The device of the present invention is particularly suitable for screening for therapeutic substances or suspected therapeutic agents produced by cells. Cell lines producing substances or therapeutic agents can also be screened with the device.

The screening can be carried out in vivo or in vitro.

 As described above, the production and screening of suspected therapeutic substances and agents is often complicated and expensive. By performing a screening procedure for a suspected substance or therapeutic agent with the present invention, the screened substance or agent is released from cells encapsulated in the device through the permeable envelope of the device, directly to a subject or to a culture under test. In using the present invention to screen suspected substances and therapeutic agents, there is no need to separate the cells from a culture medium, nor to concentrate, purify and administer the suspected therapeutic substance or agent in order to perform the screening procedure.

 In practice, a cell encapsulation device of the present invention allows a user to quickly, easily and gently transfer a desired amount of cells into the cell area of the device without having to pre-concentrate the cells. The envelope of the device is made of a material impermeable to cells, while being permeable to fluids. When an aqueous cell suspension is instilled into the cell area of the device, the cells are retained in the cell area by the envelope and the excess fluid from the cell suspension flows through the envelope of the device. This filtration of the cells by the envelope of the device continues until a desired number of cells have been placed in the cell zone. In a preferred embodiment, the envelope of the device is spontaneously wettable by liquid water and is therefore instantly ready to filter a suspension of cells when the suspension is placed in the device.

In this embodiment, when the normally opaque envelope is wetted with liquid water, it becomes substantially translucent to transparent. The envelope is substantially translucent to transparent.

The substantially translucent to transparent envelope allows a user to see into the cell area and to observe and monitor the loading of cells into the device, and the development or condition of cells in the device during use .

 The cells are transferred to the cell area of the device by a cell delivery device, such as a stainless steel syringe needle with a blunt tip, from a syringe. Other retention and distribution devices are also cell distribution devices suitable for use in the present invention; they include tissue culture pipettes, tubing from cell culture pumping devices, tubing from gravity fed cell culture vessels, etc. In addition, by using a partial or high vacuum established in the cell encapsulation device of the present invention, it may be possible to aspirate a cell suspension in the device.

 Once the cell area has been loaded with the desired number of cells, the cell delivery device is removed from the cell encapsulation device. When the cell delivery device is removed, the sealing means is actuated to contract and seal the end of the cell-encapsulating device contained therein. In the preferred embodiment, the sealing means automatically closes the opening after filling the device and / or removing the cell distribution device.

 Preferably, the device of the present invention has a nucleus which holds the encapsulated cells near or against the envelope, thereby minimizing the diffusion distances between the cells and an external source of nutrients. The core can be made of a material which may or may not swell. It is preferable to have an inflatable core and especially a water-swelling core. A particularly preferred core is one made of hydrogel material, because the hydrogel material can be initially dehydrated to reduce the size of the core, and then rehydrated to increase the size of the core. A water swellable, dehydrated core creates in the device a larger volume in the cell area than a non-dehydrated water swellable nucleus made of the same materials with the same initial dimensions.

 As a result, cells can more easily cross the enlarged cell area when the device is loaded with the cells. When the device is loaded into cells, the interval between the outer surface of the nucleus and the inner surface of the envelope of the present invention should often, however, be small. This is due to the need to minimize the diffusion distance between the cells encapsulated in the device and an external source of nutrients in order to maintain the viability of the cells. To obtain the required intervals between the nucleus and the shell, the dehydrated water-swellable nucleus is rehydrated and swollen to a predetermined size during and / or after loading the cells into the cell area. In this way, a water-swellable, dehydrated core initially provides an enlarged cell area so that cells can more easily pass through the cell area when the device is loaded with cells, and then ensures correct positioning of the cells. in the device as well as the small intervals often required for cells between the nucleus and the envelope.

 In a preferred embodiment, a dehydrated, water swellable core is rehydrated by the aqueous portion of a cell suspension when the suspension is loaded into the cell area. When the nucleus rehydrates, it swells to its size during and after the cells are loaded into the device. Other means of obtaining an inflatable nucleus may comprise an inflatable nucleus which is inflated, after placement of the cells, by introducing a gas or a liquid into it, a nucleus which is enlarged mechanically after placement of the cells, for example by axial stretching with reduction in diameter followed by return to the initial diameter, or else a polymer core which is expanded using expandable microspheres, or the like, etc. It will be noted with interest that similar results can also be obtained by providing an envelope which contracts circumferentially after placement of the cells.

 The production and assembly of the components of the device of the present invention will be described below.

 As indicated above, the core of the present invention can be made of an inflatable or non-inflatable material. Non-swellable cores can be formed from any suitable biocompatible material, such materials including polytetrafluoroethylene, poly (dimethyl siloxane) rubber, polyurethane, polyethylene, polyethylene vinyl acetate, polypropylene, polybutadiene, chloride polyvinyl, polyvinyl acetate, polyacrylonitrile, polyamide, glass or glass fibers, stainless steel, and other materials known to those skilled in the art. Poly (dimethyl siloxane) rubber is the preferred material.

The preferred core is formed from swellable polymers. Examples of swellable polymers suitable for producing the core can be chosen from the group consisting of a hydrophilic polyacrylonitrile such as the structural hydrogel HYPAN (E) (Hymedix International, Inc., Dayton,
NJ), chitin, chitosan, hydroxyethylmethacrylate (HEMA), hydrophilic polyurethane, polyethylene glycol, polyacrylamide, polyacrylic acid, and silica gel, or hydrogels derived from polysaccharides, such as alginates and other substances known to specialists in the discipline. The core is preferably a flexible polymer or elastomer or blends with copolymers of swellable and non-swellable materials. It is still preferable that the core is made from polysaccharides, hydrophilic copolymers, polyacrylonitrile, or other components. polymers. Core compositions such as the HYPANB structural hydrogel comprising a copolymer of polyacrylonitrile and acrylamide are particularly preferred.

 When a hydrogel, such as the HYPANB structural hydrogel, is used to form the core component of the present invention, the water content of the hydrated gel should be sufficient to provide flexibility while not exceeding a water content which forms pores large enough to allow cells to enter the nucleus. Preferably, the gel constituting the core is hydrated up to about 35% to 95%. With greater preference, the water content is about 80%.

 The core preferably has the general shape of a cylindrical rod which is obtained by extruding the polymer through a round die. In particular, for a core made of inflatable material, the shape of the core can be varied to take advantage of the increase in size of the core when the core is swollen. Referring to Figures 6 and 7, for example, an inflatable core can be made having additional inflatable material at one end, or both ends, of the core.

The additional inflatable material initially serves as a spacer to help maintain the dimensions of the cell area and ultimately as part of the means for sealing between the shell and the core.

 For a water-swellable core, the core material is preferably substantially dehydrated, or only partially hydrated, before assembly of the device. Preferably, the water-swellable core is dried under stress to give a rigid rod of substantially straight shape. The water swellable core rehydrates and swells when an aqueous solution is placed in contact with the core. Preferably, the water-swellable core is in the device when it is rehydrated. When rehydrated, the water-swellable core preferably swells mainly in the radial direction only. In this way, the water-swellable core has a first initial radial dimension in its dehydrated state, which is increased by inflation to a second radial dimension when the core is exposed to an aqueous solution. preferred, the water swellable core material has a first radial dimension which is at least 50% of the second radial dimension.

Optionally, a sheath having the second radial dimension desired for the core can be placed on a dehydrated core before rehydration. When the core is rehydrated, the sheath restricts the swollen core to the radial dimension of the sheath. A sheath surrounding a swollen nucleus makes it possible to form a zone of cells inside the envelope having more precise and more coherent dimensions. Among the materials suitable for the sheath, there are, without limitation, polytetrafluoroethylene, expanded polytetrafluoroethylene, polyester
Woven or microporous dacrons, woven nylon, microporous polycarbonate, woven polyacrylonitrile, Mylar @, polyethylene, polypropylene and polysulfone, alone or in combinations.

 Preferably, the aqueous solution used to rehydrate the swellable nucleus with water also contains the cells to be placed in the device. In this way, when the device is loaded with the cells, the water-swellable nucleus is rehydrated and swells to its functional size in the device. This results in optimum positioning and concentration of cells in the device.

 General information regarding the manufacture and handling of hydrogel devices can be found in the art, as in patent application PCT / US94 / 07190, from W.L.

Gore & Associates, Inc., and in U.S. Patents 4,379,874, 4,420,589, and 4,943,618, which are incorporated herein by reference.

 The selectively permeable shell component of the present invention is made of a microporous material. There is at least one opening in the envelope. Non-limiting examples of microporous polymeric materials suitable for the production of the envelope: expanded polytetrafluoroethylene (ePTFE); woven and / or non-woven polymers, such as polyester, polyethylene terephthalate, polyamide, vinyl acetates, polypropylene, polyethylene, polyacrylonitrile, polyaramide, polyhydroxy acids (such as: polylactic acid, polyglycolic acid or polycaprolactone); open cell foams such as phenolic and epoxy resins, polyurethanes, chloroprene, isoprene, polyethylene, polypropylene, polystyrene, polyvinyl chloride, latex foam rubber, polyurea-formaldehyde, polyhydroxy acids (such as: polylactic acid, polyglycolic acid, polyhydroxybutyric acid , polyhydroxyvaleric acid or polycaprolactone); and porous and / or permeable matrix membranes (such as polyvinyls, polyamide, polyimide, polyacrylonitrile, polysulfone, polyvinylidene fluoride, polypropylene, polymethylmethacrylate, polycarbonate, regenerated cellulose or cellulose acetate). These materials are perfectly usable for producing the envelope of the present invention when they are in the form of films, ribbons, sheets or tubes, for example.

The particularly preferred microporous polymeric film material is ePTFE. A biocompatible microporous metal may also be suitable for producing the envelope, either alone or in combination with microporous polymeric materials and / or surfactants or agents.
wetting agents.

Expanded PTFE, or ePTFE, is mainly characterized by a multiplicity of interconnected, open voids, defined by nodes and fibrils, high tensile strength, resistance to biological fouling and stable chemical properties. Expanded PTFE materials, including ePTFE films, can be produced in accordance with the teachings of US Pat. Nos. 3,953,566, 3,962,153, 4,096,227, 4,187,390, and 4,902,423, which are all incorporated herein by reference Films in
Expanded PTFE suitable for use in the present invention may have fibrils oriented mainly in one direction, in two directions, or in more than two directions. These films are usually referred to as uniaxially, biaxially or multiaxially oriented materials, respectively.

 Although the envelope may be made of at least one layer of a thin ribbon of microporous polymeric material, such as eE, the tensile strength and dimensional stability of the microporous polymer membrane can be varied by incorporating a or several known manufacturing techniques for producing thin films greatly increasing the tensile strength. These techniques include, without limitation, weaving or knitting filaments, laminating strips of porous films, and laminating, for example.

 An envelope of the present invention is capable of being configured in a variety of usable shapes, including, without limitation, tubes, flat sheets or ribbons, and capsules, for example. Laminating a microporous polymeric film material of the present invention is a preferred method for producing all kinds of shapes. For example, thin, highly oriented fibers or strips of microporous polymeric film can be laminated to create surfaces of varying curvature and flexibility.

 Preferably, an envelope of the present invention comprises an ePTFE material in the form of a laminate of two or more than two layers of film, oriented in different directions. This ePTFE laminate has a thickness of less than about 100 microns, preferably less than about 50 microns, and with particular preference, less than about 5 microns.

 “Laminating” is understood to mean the assembly of two or more of two layers of microporous polymer film by bringing them closely together so that the surface of one layer is very close to, and in orientation parallel with, the surface of the other. layer. The surface of a polymer film layer is considered to be very close to the surface of an adjacent parallel film layer if it is less than ten times the nominal unit thickness of the microporous polymer film constituting the laminate. Laminating microporous polymeric films can be accomplished by using the microporous polymeric film alone or in combination with an adhesive.

 The laminates produced with the microporous polymer film alone can be accomplished by intimately bonding them by heat or with a solvent. The result is a laminate of a microporous polymer film linked to the adjacent film layer by a homogeneous interaction. Homogeneous interactions include, without limitation, the creation of crystalline binding domains, glassy amorphous domains, ionic bonds, hydrogen bonds, or covalent crosslinks.

 Alternatively, an adhesive can be used to bond adjacent layers of a microporous polymeric film together, provided that the effect of the adhesive is substantially limited to the creation of bonding domains between the film layers and does not cause excessive fouling of the laminated film. Excessive fouling of the laminated film is produced when more than about 70% of the continuous passages available in the microporous polymer film are blocked. The adhesives which can be used in the present invention include, without limitation, polyurethane, polyester, and fluorinated ethylene, propylene, polyurea, polycarbonate, and polyepoxides, alone or in combination. Adhesive materials using solvents, can also be used, alone or in combination combination with the materials listed above to adhere together the layers of a microporous polymer film.

 To form an envelope of laminated ePTFE of the present invention, a film component of microporous polytetrafluoroethylene is first obtained. Such a film, having a thickness of approximately 25 microns, is a film of porous expanded polytetrafluoroethylene produced according to the teachings of US Pat. Nos. 3,953,566, 3,962,153, 4,096,227, 4,187,390, and 4,902,423, each two being incorporated herein by reference. The microporous expanded polytetrafluoroethylene film has a void volume greater than about 40%, preferably greater than approximately 75%, and with particular preference greater than approximately 85%, as measured by comparing the bulk density of the microporous expanded polytetrafluoroethylene film with a full density of non-porous polytetrafluoroethylene film. The void volume consists mainly of the passages, pores, or spaces extending through the film.

 The passages have an interval extending, in diameter or in minimum dimension orthogonal to the direction of the flow of the solute through the pore, from about 0.02 microns to about 100 microns. Alternatively, the diameter of a pore can be determined by the largest spherical particle that can pass through the pore. The passages are often interconnected inside the film. The passages which pass through the thickness of the film and open onto the two surfaces of the film form continuous passages through the film. By using the term "passages" here with reference to a microporous polymer film, it is intended to include both continuous and non-continuous passages in a particular microporous polymer film or laminate.

The void volume is related to the porosity of the microporous polytetrafluoroethylene film. The porosity index represents the space of the microporous film which does not contain film substance (i.e., the part of the film which includes pores, usually, unless the pores have been filled with some other substance (e.g. water). Expressed as a percentage, the porosity index measures the volume in percentage (%) of the microporous film which is not occupied by the microporous substance. To calculate the porosity index , in percentage, for a microporous polymer film, the following formula is generally used
Porosity = 100 (1- (p, / pf)) where:
pf is the density of the polymer material constituting the solid phase of the microporous material, and
Pa is the apparent density of the microporous film material given by the volume occupied by the outer limit of a representative sample of the microporous material divided by the mass of this material as determined by weighing the material under normal atmospheric conditions.

 The porosity index is determined for microporous polymer films, in which the empty space is empty of material, that is to say, has not been filled with any other liquid or solid impregnating the material.

 Preferably, a laminated microporous polymeric material of the present invention is produced which exhibits tensile strength in several planar directions. For example, the tensile strength of a microporous polymeric film material is usually greatest in the main direction of orientation of this film. Such oriented films generally have a tensile strength in a direction orthogonal to the main direction of orientation of such a film, and are therefore advantageously placed in a structural composite such as the main direction of orientation of such a film. is aligned with the direction of the main stress provided. To produce reinforced microporous polymeric films which exhibit tensile strength in multiple directions, the lamination of two or more layers is accomplished by having the film layers oriented in a series of distributed directions. A common two-layer laminate should include a second layer laminate having a 900 (orthogonal) orientation of the first layer. This provides a laminate called "0.90" laminate. Other lamination schemes include three-layer lamination such as "0.45, 90" laminate or "0.60, 120" laminate. A four-layer laminate could include, for example, a "0.45, 90, 135" laminate. Such multidirectional laminating schemes result in more distributed mechanical properties and less specific orientation in the final product. Alternatively, biaxially or multiaxially oriented films can be used in the present invention, either alone or in multiple layers .

 Microporous ePTFE is a hydrophobic material which does not normally allow liquid water to penetrate and pass through empty or porous spaces and passages of the material. By applying certain alcohols, liquids with low surface tension, wetting agents, or surfactants, to a microporous ePTFE material, on the other hand, the surfaces and passages of the ePTFE material can be made wettable. liquid water. For the envelopes of the present invention made of microporous ePTFE material, the normally hydrophobic ePTFE material is made hydrophilic and wettable with liquid water using alcohol, wetting agents, or hydrophilic surfactants.

Such a wettable ePTFE material allows liquid water to flow along the surfaces of the material and to penetrate and pass through the pores and passages therein. In the preferred embodiment, the envelope is spontaneously wettable with liquid water and is capable of filtering cells from an aqueous suspension.

 A method for rendering an envelope of the present invention made of ePTFE wettable with water using an alcohol, and the following method. The envelope is immersed in ethanol (at about 70 to 100%), for example, for a few seconds (about 1 to 10 s) to coat the surfaces of the material and fill the pores, or empty spaces, with l ethanol. The ePTFE material saturated with ethanol is then immersed in distilled, deionized water, or in normal physiological saline for a few minutes to displace the ethanol from the surfaces and pores of the ePTFE with liquid water. While still wet with liquid water, the envelope is loaded with cells as described below.

Although this is an acceptable method for making ePTFE wettable and usable in the present invention, some potential problems remain with this method. For example, spontaneous "dewetting" of the ePTFE envelope is a problem due to the extreme hydrophobicity of ePTFE. In addition, an ePTFE material made wettable with alcohol and then liquid water can cause air bubbles to form in the device when the device is loaded with an aqueous suspension of cells. This is believed to be due to the formation of bubbles from air trapped in the voids of the material and / or the degassing of the aqueous portion of the cell suspension wetting the ePTFE material.

 As described in more detail below, it is desirable that the envelope be substantially translucent to transparent when loading cells into the cell area of the device. Opaque ePTTE material can be made substantially translucent to transparent when wetted with liquid water using wetting agents or surfactants adsorbed on surfaces and in the void or porous spaces of ePTFE material. Therefore, ePTFE materials treated with wetting agents or surfactants are preferred materials for making the shell of the present invention.

 The problems described above associated with the fact that the ePTFE is made wettable with water with ethanol, can be avoided, or minimized, by replacing the distilled or deionized water in the process described above with a very aqueous solution. diluted with a wetting agent, such as polyvinyl alcohol. For example, 0.001% polyvinyl alcohol in saline (weight / volume) has been found to provide sufficient wetting agent to the ePTFE material to prevent, or limit, spontaneous "dewetting" of the material, to prevent or limit release of air bubbles in a device loaded with cells, and make the normally opaque ePTFE material substantially translucent to transparent. Wetting agents and / or suitable surfactants suitable for use in this process include, but are not limited to, polyvinyl alcohol, polyethylene glycol, sodium dodecyl sulfate, fluorinated surfactants, pluronics and bile salts in percentages ranging from about 0.001 to 1.0%. Solvents suitable for use in this process include, but are not limited to, saline, water, and aqueous buffers, for example.

 In the preferred embodiment of the present invention, wetting agents and / or surfactants are absorbed on the surfaces and in the voids, pores or passages, of the ePTFE envelope and are preferably immobilized in situ in order to make the material wettable with liquid water. There are several ways to immobilize wetting agents or surfactants, such as crosslinking, grafting on substrate, immobilization by plasma, ionic complexing and grafting of free radicals, etc. In one example, by crosslinking the wetting agent or surfactant on the ePTFE in situ, the wetting agent or surfactant is immobilized on the ePTFE material. Some wetting agents or surfactants can be used, which make the ePTFE spontaneously and substantially completely wettable with liquid water. An ePTFE material spontaneously and substantially completely wettable with water allows liquid water to flow along the surface and through the passages of the material by simple contact of the material with liquid water. Wetting agents or surfactants suitable for use in the present invention include, without limitation, polyvinyl alcohol, poly (tetrafluoroethylene-co-vinyl alcohol), polyacrylic acid, polyethyleneimine, and polyethylene glycol .Wetting agents and / or surfactants are adsorbed in various ways, such as in solution, or undiluted, by adsorption, vapor deposition, immobilization by plasma, and thin film assembly, for example. Preferably, the alcohol is adsorbed on ePTFE by adsorption of polyvinyl alcohol on the surfaces and in the empty or porous spaces of the material then by immobilization by crosslinking of the polyvinyl alcohol to itself with a dialdehyde such as glutaraldehyde.

 An envelope of the present invention made of water-wettable ePTFE is strong enough to withstand sufficient hydrostatic pressures to force water through the pores of the material through the thickness of the envelope. When water is forced through the thickness of the envelope, the water-wettable ePTFE material functions as a filter, or an ultrafilter, depending on the permeability of the material. As water moves, or infiltrates through the thickness of the envelope, it tends to collect in droplets on the outer surface of the envelope. When adjacent droplets grow in size, they merge and run over the envelope. This process is referred to herein as "exudation". Most of the water-wettable casings of the present invention are sufficiently permeable to water that pressurized water visibly exudes from the casing without forming much grooves .

 Ideally, the envelope of the present invention is sufficiently permeable to water to allow water to easily separate from the cells under relatively low pressure. A formed exudation flow between about 0.01 ml / cm2 / minute and about 100 ml / cm2 / minute at a pressure between less than about 3.4 X 104 Pa and about 6.9 X 105 Pa must allow a relatively rapid concentration of cells inside the device.

 This is an extremely advantageous feature of the present invention. Unlike previous devices containing cells which require a concentration of cells before introducing them into the device, and then a carefully calculated and controlled transfer, the present invention allows the cells to be easily transferred to any desired concentration with steps of pre- minimum concentrations. In addition, by flushing clear water into the cell-filled apparatus after the initial cell loading, a user can ensure that the cells are indeed carried by the water into the device and not left on the device as a spoiled residue.

 Another advantage of the envelope becoming substantially translucent to transparent is that, in a translucent or transparent state, the cells in the device can be observed through the envelope both during and after loading of the cells. This not only helps with cell loading, but also makes cell monitoring much easier during use.

The selectively permeable envelope must have a pore size range sufficient to prevent movement of cells inside or outside the device, but large enough to allow passage of nutrients, waste, and substances therapeutic secreted by the cells contained in the device. In one embodiment, the characteristic pore size is small enough to filter or exclude particles on a molecular scale. Such molecular weight limit value (MWCO) properties can be useful in preventing proteins, etc., produced by a recipient's immune system from crossing the envelope and adversely affecting cells encapsulated in the device. 'interval
Precise MWCO will vary depending on the material of the membrane, the type of cells in the device, the size of the cell's therapeutic product to be released into the surrounding environment, and the host environment, etc. Under these conditions, a selectively permeable envelope having an MWCO of between about 10 kD and about 2000 kD may be suitable for use in the present invention. Interval
MWCO of between approximately 30 kD and 500 kD is particularly advantageous in applications where it is desired to isolate the contained cells from contact with the molecules of the immune system capable of recognizing and destroying the contained cells.

 Referring to Figure 1, we will describe below an advantageous method for assembling a device according to the present invention. A core 11, substantially dehydrated or only partially hydrated, and a selectively permeable envelope 12, of tubular shape, are prepared separately. The envelope 12 has an opening at each end. The core 11 is placed in the envelope 12 and is retained there by a sealing means 16 in the substantially dehydrated state or only partially hydrated until the cell zone 14 is loaded with an aqueous suspension of cells.

 Before placing the core 11 in the envelope 12, a spacer 18, preferably in the form of a ring, is optionally placed on one end of the core 11. The luminal surface of the spacer 18 is preferably formed to be conform to the shape of the external surface of the core 11. The external surface of the spacer 18 is preferably formed to conform to the luminal surface of the envelope 12.

 The spacer 18 fulfills various functions in the present invention, which depend on whether the core 11 is made of an inflatable or non-swellable polymer. For a core 11 made of non-inflatable polymer, the spacer 18 maintains the radial dimension of the cell zone 14 at the end of the device, while providing a structure against which a sealing means 16 acts to tighten and seal the envelope 12. For a core 11 made of inflatable polymer, the spacer 18 maintains the sealing means 16 in a stretched conformation until the inflatable core is rehydrated and swells from a first radial dimension to a second radial dimension. Once the inflatable core has been rehydrated and inflated, the part of the device containing the spacer is preferably removed. The removal of this part of the device is conveniently carried out with a sharp edge, such as a scalpel blade, at point "A" in FIG. 1, for example. The point "A" in FIG. 1 indicates, in general, the surface in which a cut is optionally made in the device 10 for removing the part of the device containing the spacer 18, as well as the corresponding part of the envelope 12 , and the corresponding part of the core 11.

 Once the part of the device containing the spacer has been removed, the sealing means 16 tightens, to make it watertight, the envelope 12 against the swollen core 11 (see FIG. 4, for example). It should be understood, however, that removal of the portion of the device containing the spacer 18 is not necessary for the device to be sealed, if the sealing means provides a fluid-tight seal between the outer surface of the swollen core 11 and the internal surface of the spacer 18.

 Once the spacer 18 has been applied to the core 11, the core is inserted into the envelope 12, as shown in FIG. 1. A sealing means 16, such as a constriction device, is then placed on the envelope 12 to compress the envelope against the spacer 18, thereby securing the core 11 at the end of the envelope 12. If the spacer 18 is not applied to the core 11, the core 11 is inserted into the envelope 12 and a sealing means 16 is placed on the envelope 12 and can tighten the envelope 12 against the core 11, thereby securing the core 11 at the end of the device 10 and also sealing this end.

 Materials suitable for the spacer 18 include, without limitation, polytetrafluoroethylene, polypropylene, polyethylene, poly (dimethyl siloxane) rubber, mink, buton, and elastomers, hydrogel materials such as hydrogels structural HYPANS (Hymedix International, Inc., Dayton, NJ), hydroxyethylmethacrylate, and polyvinyl acetate, biocompatible metals, such as stainless steel and titanium, for example.

 As illustrated in Figures 6 and 7, the spacer can optionally be replaced or reduced in size, with additional core material on one end of the core, or on both ends. The assembly of a device of the present invention having a core as illustrated in Figure 6 is similar to the assembly of a device having a spacer at one end of the core.

 An envelope having only one open end is treated as an envelope having two open ends, except that the core, optionally having a spacer in place or additional core material at one end of the core, is inserted into the envelope through the opening of the device to the side of the device opposite to the opening as illustrated in FIG. 2. A sealing means 26 is placed over the casing 22, the optional spacer 28 or the additional core material, and the core 21 for compressing the casing 22 against the spacer 28 or the additional core material, thereby securing the core to the device. Alternatively, the spacer 28 or the additional core material can be eliminated and the sealing means 26 used to compress the casing 22 directly against the core 21 to seal the device 20.

 Referring to Figure 1, the remaining open end of the device is assembled with a cell delivery device 19 attached to the cell encapsulating portion of the present invention by sealing means 16, or a device Tightening. Preferably, the cell delivery device 19 is a blunt-tipped syringe needle made of stainless steel or an equivalent material. Other suitable cell delivery devices include, but are not limited to, cannulas, cylindrical tubes, flattened tubes, tissue culture pipettes, tubing connected to cell culture pumping devices, and tubing connected to containers of cell cultures fed by gravity, etc. The materials suitable for producing a cell distribution device include, without limitation, polytetrafluoroethylene, poly (dimethyl siloxane) rubber, polyurethane, polycarbonate, polyethylene, polyethylene vinyl acetate, polysulfone, polypropylene, polybutadiene, polyvinyl chloride, polyvinyl acetate, polyamide, glass or glass fibers, stainless steel, etc.

 In embodiments having a nucleus as illustrated in Figures 1, 2 and 6, for example, the diameter of the cell delivery device must be sufficiently greater than the outer diameter of the nucleus for cells to be introduced into the cell area of the device without being subjected to sufficiently large shear forces to damage them. The outer diameter of the needle must be smaller than the inner diameter of the envelope so that the envelope can be placed on the end of the needle.

 The unclosed end of the core is placed in the dispensing end of the needle as shown in Figures 1, 2, and 6, for example. The unclosed end of the envelope is placed over the outer surface of the needle delivery end as shown in Figures 1, 2, and 6, for example.

 In the embodiments having a core as illustrated in FIG. 7, for example, the delivery end of the cell delivery device is placed in the envelope and adjacent to the core as shown in FIG. 7.

 In the embodiments illustrated in Figures 1, 2, 6, and 7, for example, a sealing means is placed both on the envelope and on the end of the needle of the cell delivery device thus securing the cell encapsulation portion of the device to the cell delivery device. The sealing means preferably comprises a sleeve of polymer material having elastomeric properties. The sealing core is also called here "clamping device".

 The "sealing means" as used herein refers to devices or materials which serve to provide a seal between the shell and a spacer, or in the absence of a spacer, between the shell and the core .

Certain sealing means provide a circumferential clamping force on the envelope, pressing it against the spacer or the core. Examples of such sealing means include, without limitation, elastomeric clamping rings, fibers or films wound on themselves, heat activated clamping tubes, and chemically activated clamping tubes, by example. Mechanical collars, such as those based on plastic deformation of a strip (for example, embossing of a metal or plastic strip), pipe clamps, band clamps, crimping, and pipe clamps flat bars are also considered as sealing means. Other sealing means provide sealing by expanding from the inside of the device towards the outside against the envelope.

For example, an inflatable core can inflate and expand radially to reach the internal dimensions of the envelope, thus forming a seal. With this method, it is preferable to reinforce the envelope so that it resists subsequent swelling of the core from the interior of the device in order to form a seal. A core can be confined, or prestressed to a radial diameter smaller than the internal diameter of the envelope. Once the core is placed in the envelope, the core is released from confinement and can expand by pressing closely against the envelope to form a seal by interaction, or by adjustment. The device of the present invention can also be made waterproof with an adhesive, by heat, by ultrasound, etc.

 One method of sealing the device with adhesive is to immerse one end of the core in the adhesive and to position the end coated with adhesive in the casing. Then finishing steps appropriate to the particular adhesive are carried out in order to seal the device.

 Suitable materials for making clamping devices, or sealing means, include, without limitation, poly (dimethyl siloxane) rubber, fluoroelastomers, urethanes, and hydrogels. Poly (dimethyl siloxane) rubber is the preferred material.

 The preferred materials for making mechanical collars include, without limitation, stainless steel, titanium, nylon, polysulfone, engineering plastics, and composites of these materials.

 Adhesives suitable for sealing the device include, but are not limited to, cyanoacrylate, poly (dimethyl siloxane) rubber, urethane, and hydrogels.

 Alternatively, the sealing means may consist of an interpenetrating network of an ePTFE envelope material and an elastomeric polymer. For example, uncured poly (dimethyl siloxane) rubber can be applied to surfaces and impregnated in voids, or porous spaces, with an ePTFE shell material on the envelope areas to be sealed. After the assembly of the core and the envelope as described above, a clamping device is placed on the areas of the envelope having the interpenetrating network described above. The poly (dimethyl siloxane) rubber is then hardened to seal the junction of the shell and the core. The clamping device is usually removed once the seal has been formed, ideally this process produces a seal with few or no creases in the envelope. Although not likely to be critical, creases in the envelope at the sealing site are undesirable because creases can create areas in which cells can be trapped or which can lead to cell leakage. .

 Preferably, the device is made sterile before use. In one embodiment, each component of the present invention is sterilized before assembly of the device. The assembly of the present invention is preferably carried out under aseptic conditions. In another embodiment, a device of the present invention is sterilized after assembly. Suitable sterilization methods include, but are not limited to, gamma, ultraviolet, or other radiation, dry heat, ethylene oxide, and water vapor, alone or in combination. Steam sterilization is preferred.

 In practice, a sterilized device of the present invention is attached to an aseptic syringe containing a suspension of cells to be loaded into the device, as shown in Figure 3. This procedure is performed under sterile conditions. As illustrated in Figure 5, the syringe is then gently actuated to transfer the cell suspension to the cell area 54 of the device 50 under relatively low pressure (e.g. from about 7 kPa to about 140 kPa) through the device when distributing cells 59. When the cell suspension is introduced into the cell zone 54, the cells begin to fill the cell zone 54 while the aqueous part of the suspension comes into contact with the nucleus 51 and the envelope 52 When it comes into contact with the aqueous part of the cell suspension, the nucleus begins to rehydrate and expand from a first initial radial dimension to a second enlarged radial dimension. For most applications it is preferable that the dehydrated hydrogel core swells minimally in a longitudinal direction when hydrated. Substantially at the same time, the envelope spontaneously and substantially completely wets with liquid water. The normally opaque envelope made of ePTFE treated with a wetting agent or a surfactant, becomes substantially translucent to transparent when it is wetted with liquid water. as a result, the loading of cells into the device can be observed and monitored visually through the envelope.

 The loading of cells into the device continues until the desired concentration of cells is achieved. In some applications, the cells are sparingly loaded in the device and allowed to grow in the device. In other applications, cells are loaded at high densities into the device so as to substantially fill the device with cells. Due to the suitability of the shell 52 for exudation, additional cell suspension can be introduced into the cell area 54 of the device without causing the device to overflow or burst with the pressurized liquid component of the suspension cells. As a result, the envelope can exude as much as needed while the cells are loaded into the device to the desired concentration.

 When the device 50 is filled to the desired concentration of cells, the device is separated from the cell delivery device 59, as shown in Figure 4. During this process, the open end of the device through which the cells have been loaded is closed automatically with the sealing means 56. In order to separate the device 50 from the cell distribution device 59, the cell distribution device is removed from the envelope 52 of the sealing means 56. When the delivery device distribution of cells 59 is removed from the sealing means 56, the sealing means, or clamping device, is actuated to tighten the envelope 52 against the core 51 thereby hermetically closing the open end of the device and encapsulating the cells contained therein.

 When the loading of the cells is completed and the cell distribution device 59 is removed from the device 50, the sealing means 56, part of the casing 52, and part of the core 51 can be removed by cutting. considered end of the device, to ensure that all cells left in the open end of the device are eliminated. The cut end of the device can be cleaned or made sterile by various means, such as rapid washing of the end with ethanol solution, brief contact of the end with a heated instrument, or by immersion of the end in deionized or distilled water. The cell encapsulation device with its cell reserve can then be cultured in vitro or implanted in vivo.

 Although this description mentions that the present invention is particularly suitable for cells encapsulated for screening suspected therapeutic substances and for gene therapy, there are many uses for the present invention in which cell encapsulation is desired. Cells suitable for use in the present invention include, but are not limited to, eukaryotic cells, such as cells which are autologous to a recipient, cells which are allogenic to a recipient, cells which are xenogenic to a recipient, gametes, and prokaryotic cells.

 Without wishing to limit the scope of the present invention, the following examples illustrate how the present invention can be made and used.

EXAMPLE 1
An envelope of the present invention has been made in the form of a tube having a microporous polymer film component consisting of multiple layers of an expanded polytetrafluoroethylene (ePTFE) film laminated oriented together in multiple directions, relative to the main orientation axis of each ePTFE film layer.

 An ePTFE film was used to make a laminated tube about 5 cm in length. The microporous film was made in accordance with U.S. Patent No. 3,953,566 as follows and with the following properties. The film was first elongated in a single direction to form fibrils oriented mainly in the direction of elongation to produce an oriented film. This film was characterized by a tensile strength of 6.9 X 104 Pa (10,000 psi), a porosity of approximately (16 Gurley seconds, a width of approximately 6.0 mm, and a thickness of approximately 28.0 microns, as determined with a laser micrometer The first of the two film layers was applied to a 1.6 mm mandrel, the film fibrils being oriented longitudinally with respect to the axis of the composite membrane. The second film layer was wrapped over the first film layer at an angle of about 60 from the first film layer with a slight overlap of about 2 mm occurring on each successive helical winding. heat welded in a convection oven heated to 3800C for about 5 minutes The resulting envelope was resistant in all directions, retained its shape well, and was able to filter cells.

EXAMPLE 2
This example illustrates the optional step making the ePTFE material of Example 1 spontaneously and substantially completely wettable with water by absorption and crosslinking of a hydrophilic fluoropolymer, poly (tetrafluoroethylene-co-vinyl alcohol) (HPL), in the microporous voids of the ePTFE material and on the surfaces of the material.

 The tubular ePTFE laminate described in Example 1 was immersed in 95% ethanol for about one minute to coat the surfaces and almost completely fill the microporous voids of the material with ethanol. The ePTFE material was removed from the ethanol solution and immediately placed in a solution of 1.5% (w / w) HPL in methanol: ethanol (4: 1) for about 5 minutes. Care was taken not to let the ePTFE material dry out while transferring the material from one solution to another.

 The ePTFE material was then removed from the HPL solution and blot-dried with paper towels or the like to remove excess solution. The eFTFE material, with adsorbed HPL, was immersed in methanol for about 5 minutes, removed, blotted, and again immersed in methanol for about 5 minutes to wash away any free HPL. Care was taken not to allow the ePTFE material to dry out between transfers in the methanol solutions.

 The absorbed HPL ePTFE material, rinsed with methanol, was further rinsed in deionized water for about 5 minutes. As before, the ePTFE material was not allowed to dry out between the transfer of the material from the methanol solution to deionized water.

 The HPL copolymer impregnated in the microporous voids and covering the surfaces of the ePTFE material was fixed in situ by crosslinking the copolymer with glutaraldehyde. After removing the ePTFE material from the deionized water rinse bath, the material was gently blotted and immediately placed in a preheated aqueous solution (i.e., about 80 "C) at 5% (v / v) glutaraldehyde ( EM grade) with 1% (v / v) HCl as catalyst The crosslinking reaction was allowed to develop for about 5 minutes at about 80 "C. After the crosslinking reaction, the ePTFE material was removed from the glutaraldehyde solution and immersed in deionized water for about 10 minutes to rinse off any free glutaraldehyde.

 The ePTFE material treated with hydrophilic fluoropolymer was then air dried and steam sterilized.

EXAMPLE 3
This example and similar to Example 2 except that polyvinyl alcohol was used as a hydrophilic polymeric wetting agent in place of HPL.

 In this process, the ePTFE material of Example 1 was immersed in about 95% isopropanol for about 0.5 minutes to cover the surfaces and substantially completely fill the microporous voids of the ePTFE material with isopropanol. Care has been taken to ensure that no air bubbles cling to the ePTFE material during this process.

 The ePTFE material was removed from the isopropanol solution, gently blotted, and immersed in an aqueous solution of 1% polyvinyl alcohol (v / v) which was 100% hydrolyzed, having a molecular weight of about 90 kg / mol. The ePTFE material was subjected to the polyvinyl alcohol solution for about 5 minutes. The ePTFE material was not allowed to dry out during the transfer between the isopropanol solution and the polyvinyl alcohol solution. The ePTFE material was then removed from the polyvinyl alcohol solution, blot-dried, and immersed in deionized water for about 10 minutes to rinse off any free polyvinyl alcohol. The ePTFE material was not allowed to dry out during the transfer between the polyvinyl alcohol solution and the deionized water.

 The polyvinyl alcohol surfactant adsorbed in the microporous voids and covering the surfaces of the ePTFE material was fixed in situ by crosslinking the surfactant with glutaraldehyde. After removal of the ePTFE material from the deionized water rinse bath, the material was gently blotted, and immediately placed in an aqueous solution of 1 to 5% (v / v) of glutaraldehyde (EM quality) and 1% (v / v) HCl for approximately 2 minutes.

 After the crosslinking reaction, the ePTFE material was removed from the glutaraldehyde solution and immersed in deionized water for about 10 minutes to rinse off any free glutaraldehyde.

 The ePTFE material treated with a hydrophilic polymeric wetting agent was then air dried and steam sterilized.

EXAMPLE 4
A device of the present invention was aseptically assembled as follows. As described above, all components of the device are sterilized before assembly. Preferably, the components are steam sterilized. The hydrogel core, for example, was steam sterilized in the hydrated state and then stress-dried in a sterile package.

 Referring to Figure 1, a dehydrated hydrogel core 12, having nominal dimensions of about 0.75 mm outside diameter (OD) in the dehydrated state and about 1.4 mm from OD to l hydrated state, was inserted into a tubular casing 12 of Example 3 having a wall thickness of 50 μm, an average pore size of 0.3 μm, and a porosity measured according to the Gurley procedure of approximately 20 seconds, a nominal OD of approximately 1.62 mm with openings at both ends of the tube.

A spacer 18 in the form of a ring made of poly (dimethyl siloxane) rubber with a nominal OD of about 1.19 mm and a nominal internal diameter (ID) of about 0.64 mm, was placed on one end of the core about 5 mm from the end. The core and spacer were inserted into the shell, one end of the core projecting approximately 5 mm beyond the end of the shell. A sealing means 16 in the form of a silicone clamping ring, about 10 mm in length, was placed around the envelope, as shown in Figure 1, and allowed to tighten. envelope against the sealing means.

 A cell delivery device 19 in the form of a sixteen gauge (16) hypodermic syringe made of stainless steel with a blunt end, 3.81 cm long, was inserted into the opposite end of the tubular casing 12 The needle was placed in the envelope so that the core 11 located inside the envelope is inserted into the open blunt end of the needle. The needle was inserted into the envelope, over the core, so that the hypodermic needle penetrates about 5 mm into the lumen of the envelope.

 A sealing means 16, in the form of a silicone rubber tightening ring, of approximately 5 mm in length, was placed on the end of the envelope comprising the hypodermic needle placed in it in such a way that the silicone clamping ring surrounds the end of the envelope and the end of the hypodermic needle.

When the silicone clamping ring was left to act, it tightened the envelope 12, in a leaktight manner, against the cell distribution device 19.

EXAMPLE 5
Cells were loaded into the device of Example 4 without spreading the cells or extrinsically contaminating the device, as follows. Two cell suspensions were loaded into a device of the present invention in this example in order to show how to operate a monitored and controlled cell loading in the device. Sterilized items were used: instruments, glassware, plastic objects, cell culture media and diluents during the loading of the device into cells.

 A cell suspension was prepared in a volume of culture medium which was approximately double the volume of the device, plus any non-recoverable volume associated with the cell delivery device. 300 µl of cell suspension was prepared in the present example. Once prepared, the cell suspension was placed in a 1 ml syringe. The syringe has been purged of any air bubbles. A second volume of 300 µl of cell suspension was similarly prepared in a 1 ml syringe.

 A large hemostatic forceps, or the like, was used to grip and hold the hypodermic needle near the silicone rubber seal, but not on the device shell. After positioning the device in a generally horizontal orientation, the 1 ml syringe containing the cell suspension was connected to the hypodermic needle. The syringe plunger was actuated slowly to transfer the contents of the syringe into the cell area of the device.

 When the contents of the syringe entered the cell area of the device, the water-swellable, dehydrated nucleus began to rehydrate and swell in diameter. The envelope of the device spontaneously wetted with liquid water and went from an opaque to a transparent appearance when it came into contact with the contents of the syringe. When the device was filled with the contents of the syringe, the culture medium began to filter, or "exude" through the walls of the envelope. Care was taken during this process not to overpress the device to the point of leakage of the cell suspension through the sealing means.

 Once the results of this first injection of cells into the device are noted and considered acceptable, the syringe containing the first suspension of cells has been removed from the hypodermic needle component of the device and the syringe containing the second suspension of cells has been attached to the hypodermic needle in its place.

 Care was taken to prevent any air bubbles from the tip of the syringe or the end of the hypodermic needle from being drawn into the fluid circuit.

 A second injection of cells into the cell area of the device was then performed as for the first injection. The process was monitored through the transparent envelope of the device until the device was filled with the desired amount of cells.

 After the cell area was filled with cells, the syringe was disconnected from the Luer connection portion of the hypodermic needle and a Luer obturator was inserted in place of the syringe. The end of the Luer obturator needle was immersed in deionized water to rinse and kill any cells that may have escaped from this junction. The envelope of the device was not allowed to come into contact with deionized water.

 The whole device was then immersed in sterile culture medium in a petri dish to a depth of about 4 mm and incubated at 37 "C for about an hour. Meanwhile, the hydrogel core continued to swell to its final external diameter The swelling of the nucleus has optimally positioned the cells encapsulated in the device for maximum viability and productivity.

 At the end of the device comprising the spacer, the external surface of the fully inflated core and the internal surface of the envelope were in substantial or close contact in the region of the sealing means at this time. To complete the sealing process at this end of the device, the end of the device comprising the spacer was cut to remove the part of the device containing the spacer and the corresponding parts of the casing and of the core. This had the consequence that the sealing means 46 tightened the envelope 42 against the core 41 as illustrated in FIG. 4.

 The closed hypodermic needle was removed from the cell encapsulation portion of the device by first gripping the hypodermic needle near the silicone sealant, but not on the envelope, with a large hemostatic forceps. The Luer obturator was removed from the hypodermic needle to discharge the assembly. Using a rubber foot forceps, or the like, the device was gripped on the silicone seal ring near the hypodermic needle at a point very close to where the blunt end of the needle enters in the sealing ring. The device was positioned in a substantially horizontal orientation on a sterile dry field when the needle was slowly withdrawn from the cell encapsulation part of the device with the large hemostatic forceps. When the needle was withdrawn from the part d Once the cells of the device were encapsulated, the silicone sealing ring tightened automatically and applied the envelope tightly to the core of the device. During this part of the process, care was taken to confine any drop that emerges from the device at the end of the device near the seal and not to let any drop reach the envelope of the device.

 The recently sealed end of the cell encapsulation part of the device has been carefully cut with scissors to ensure an airtight seal and prepare the end for sterilization to kill or remove any cells that may have escape through the loading site. The cut end provided a silicone seal ring having a length of about 10 mm.

 Once the two ends of the device were cut, the sealed ends of the device were immersed in deionized water for about 20 seconds to kill any escaped cells. Care was taken to avoid placing the envelope portion of the device in deionized water.

 To remove any cellular contaminants on the outside of the device that might have been present after loading, the cell encapsulation device, taken in isolation, was rinsed for several seconds in Hank's balanced saline solution (HBSS).

 The device was then placed in cell culture medium in a petri dish to a depth of about 4 mm and cultured at 370C for at least two hours before use.

EXAMPLE 6
Cells encapsulated in devices of Example 5 were cultured in vitro as follows. Two devices were filled with rat insulinoma cells, cells (RIN). Two other devices were filled with CGT-6 cells. Both devices containing RIN cells were cultured in media
RPMI containing 10% fetal bovine serum. The two devices containing CGT-6 cells were cultured in OP media
DMEM. Each device culture was made in a T-25 tissue culture flask having a volume of about 4 ml of medium. The devices containing cells were cultured for 17 days. The media were changed daily in each culture flask. At the end of the culture period, the devices were briefly examined for cell viability. Each device contained a population of viable cells after an in vitro culture for more than two weeks.

EXAMPLE 7
Devices of Example 5 were implanted in each of three naked rats, as follows. Each naked rat received two devices, subcutaneously, on each side of the peritoneal cavity. Each device was loaded with approximately 1 X 105 rat insulinoma cells, cells (RIN).

 At two weeks, the devices were explanted from rats and the cells encapsulated in the devices were tested for viability.

Using a neutral red tint, it was determined that the cells of the device were viable.

 While particular embodiments of the present invention have been illustrated and described herein, the present invention should not be limited to such illustrations and descriptions. Obviously, changes and modifications may be incorporated as part of the present invention within the scope of the claims.

Claims (19)

 1 - Cell encapsulation device comprising:  a selectively permeable shell comprising a microporous material;  a core within the shell, the core comprising an inflatable material which expands from an initial first radial dimension to a second enlarged radial dimension upon exposure to an aqueous solution;  at least one opening in the envelope,  a cell delivery device for transferring cells into the device through the opening; and  a means for sealing the opening after the cells have been introduced into the device.  2 - cell encapsulation device according to claim 1, wherein the cell delivery device comprises a tube mounted in the opening adapted to transfer cells from a separate container in the device.  3 - cell encapsulation device according to claim 1, wherein the means for sealing the opening comprises a clamping device to the opening which contracts to automatically seal the opening when removed the opening cell dispensing device.  4 - cell encapsulation device according to claim 1, wherein the inflatable material has a first radial dimension which is at least 50% of the second radial dimension.  5 - cell encapsulation device according to claim 1, wherein the envelope comprises a wettable material which becomes essentially translucent to transparent by introduction of an aqueous solution into the device.  6 - cell encapsulation device according to claim 1, wherein the device comprises means for filtering the cells from the solution, this means comprising the formation of the envelope with a material which is permeable to water and impermeable to cells and whereby by introducing cells and solution into the pressurized device, the solution exudes through the envelope and the cells remain inside the device.  7 - cell encapsulation device according to claim 1, wherein the envelope comprises at least one layer of a thin ribbon of expanded polytetrafluoroethylene.  8 - cell encapsulation device according to claim 1, wherein cells are contained in the device.  9 - Method for loading cells into a cell encapsulation device which comprises  providing a cell encapsulation device comprising a selectively permeable envelope having at least one opening, the envelope being permeable to water but impermeable to cells  providing a cell delivery device for transferring an aqueous solution of cells into the device from a separate container containing cells;  transferring the cells from the separate container to the cell encapsulation device under pressure; and  allowing water to exude from the envelope during cell transfer, concentrating the cells in the device.  10 - The method of claim 9 further comprising providing a cell encapsulation device comprising an inflatable core; and  swelling of the nucleus after the cells are transferred to the device.  11 - The method of claim 9, further comprising  providing an opening clamp to seal the opening of the device; and  actuation of the clamping device to seal the opening after the transfer of cells into the device.  12 - Cell encapsulation device comprising  a selectively permeable envelope comprising a microporous water-permeable and cell-impermeable material and by which, by introducing cells and solution into the pressure device, the solution exudes through the envelope and the cells remain in the device;  a core in the shell;  at least one opening in the envelope;  a cell delivery device for transferring cells into the device through the opening; and  a means for sealing the opening after the cells have been introduced into the device.  13 - cell encapsulation device according to claim 12, wherein the nucleus comprises a non-swellable material.  14 - cell encapsulation device according to claim 12, wherein the nucleus comprises an inflatable material.  15 - Cell encapsulation device according to claim 12, wherein the cell delivery device comprises a tube mounted in the opening adapted to transfer cells from a separate container in the device.  16 - cell encapsulation device according to claim 12, wherein the sealing means of the opening comprises an opening clamping device which contracts to automatically close the opening sealingly when the opening cell delivery device.  17 - cell encapsulation device according to claim 12, wherein the envelope comprises a wettable material which becomes essentially translucent to transparent by introduction of an aqueous solution into the device.  18 - Device for encapsulating cells according to claim 12, in which the envelope comprises at least one layer of a thin ribbon of expanded polytetrafluoroethylene.  19 - Cell encapsulation device according to claim 12, in which cells are contained in the device.